The invention relates generally to compositions and methods that are effective in inducing apoptotic cell death and inhibiting replication and repair in normal, abnormal or cancerous cells.
Apoptosis or programmed cell death, is an essential physiological process required for development, homeostasis and protection by the immune system (Thompson, C. B., 1995, Science 267: 1456-1462). It is a property of animal cells, allowing unwanted cells to be eliminated quickly and neatly. Although the death program is cell-intrinsic, it is regulated by extracellular signals that can either activate it or suppress it (Raff, M. C., 1992, Nature London 356: 397-400). The dependence on survival signals ensures that a cell only survives when and where it is needed, just as dependence on growth factors for proliferation ensures that a cell only divides when a new cell is needed. The importance of such controls in multicellular organisms is illustrated by the devastating effects of proliferative diseases, inflammatory diseases, arteriosclerosis or cancer, where the controls are defective. Furthermore, attenuation of apoptotic potential is associated with cancer progression and resistance to chemotherapy. Some chemotherapeutics and other relevant cancer, anti-proliferative and anti-inflammatory therapies induce apoptosis in their targets and apoptosis resistance contributes to metastasis of some cancers such as prostate cancer (McConkey, D. J. et al., 1996, Cancer Res. 56:5594-5595). Therefore, agents that are effective inducers of apoptosis are needed to implement effective apoptosis and therefore, effective prevention and progression of proliferative diseases, cancer, arteriosclerosis and inflammatory diseases. Cytoplasmic shrinkage, chromatin condensation, and fragmentation of DNA are widely accepted distinguishing features of apoptotic cells. In contrast, necrotic cells typically display cytoplasmic swelling and lysis of the cell membrane and do not exhibit the condensed and fragmented chromatin associated with apoptosis.
The present invention demonstrates agents which induce apoptosis as well as modulate the activity of proliferating cell nuclear antigen (PCNA) which results in inhibition of replicative DNA synthesis. Eurkaryotes contain six DNA polymerases including DNA polymerases .epsilon. and .delta. which are essential for replicative DNA synthesis. PCNA was identified as a specific auxiliary factor of polymerase .delta. and to stimulate polymerase .epsilon., implying that PCNA interacts with both polymerases and modulates replicative DNA synthesis (Tan, C. K. et al., 1986, J. Biol. Chem 261: 12310-12326; and Yoder, B. L. et al., 1991, J. Biol. Chem 266: 22689-22697). Therefore, agents that modulate PCNA activity which results in inhibition of replicative DNA synthesis are generally effective in inhibiting proliferative diseases, cancer cell development and proliferation, arteriosclerosis and inflammatory diseases.
The present invention is directed to compositions and methods using organic acid and inorganic acid salts of 5-amino-imidazole carboxamide (AICA) and/or of 5-amino or substituted amino, 1,2,3-triazoles (triazoles). For example, AICA and/or triazoles may be reacted with: a) aliphatic acids including, but not limited to, lactic, succinic, maleic, citric, tartaric or orotic; b) sugar acids including, but not limited to, gluconic or galactonic; or c) inorganic acids including, but not limited to, hydrochloric acid and phosphoric acid, to form salts suitable for use according to the compositions and methods of the present invention.
AICA salts have been used as hepatoprotectants based on their ability to prevent necrosis and stimulate regeneration of liver parenchymal cells. AICA orotate was also found useful in the inhibition of experimental prostatic cancer in rats (Cohen, L., Wehrmann, F. and Karmali, R. Proc. Am. Assoc. Cancer Res. 38:607).
Triazoles were originally disclosed as having anticoccidial activity in poultry (U.S. Pat. No. 4,590,201, issued May 20, 1986), and later in the treatment of peritoneal carcinomatosis of ovarian cancer (U.S. Pat. No. 4,132,315, issued Jul. 21, 1992 and Kohn, E. C et al., 1990, J. Natl. Cancer Inst. 82: 54-60), and of the PMT-6 fibrosarcoma tumor model in mice (U.S. Pat. No. 5,045,543, issued Sep. 3, 1991). One triazole compound in particular, the 5-amino-1-(4-[4-chlorobenzoyl]-3,5-dichlorobenzyl-1,2,3-triazole-4-carboxa mide, designated L651582 (Merck Research laboratories, U.S. Pat. No. 4,590,201) was shown to inhibit cell proliferation, inflammation and some signal transduction pathways including those which involve calcium influx, the release of arachidonic and the generation of inositol phosphates (Kohn, E. C. et al., 1992, Cancer Res. 52: 3208-3212; and Felder et al., 1991, J. Pharmacol. Exp. Ther. 257: 967-971). Furthermore, the orotate salt of L651582 was shown to have a greater inhibitory effect on prostatic tumors in rats (Cohen, L., Wehrmann, F. and Karmali, R. Proc. Am. Assoc. Cancer Res. 38:607).